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Samples were stained with Mayer's hematoxylin (MHS-1, Sigma Aldrich) and 1% eosin in CaCl2 (318906, Sigma Aldrich) following a series of alcohol-mediated dehydration steps, and stained sections were mounted in clarion mounting medium C-04877, SigmAldrichch).

A major advantage of this method compared to the usual techniques is the lack of purification steps and staining procedures, which often lead to cell activation.

Following washing steps and staining with Streptavidin-Cy3, arrays were scanned on the Illumina® BeadArray Reader and the images analyzed using the Illumina BeadStudio software.

After washing, the membranes were incubated for 1 h with anti-DIG antibody conjugated with ALP (1 1000 in TBS), which was followed by three wash steps and staining with NBT/BCIP (1 50 in 0.1 M Tris-HCl, 0.05 M MgCl2 and 0.1 M NaCl at pH 9.5).

In the experiments determining the expression of C5L2 on mouse peripheral neutrophils, purified anti-C5L2/GPR77 anti-C5L2/GPR77R&D Systems, Wiesbaden-Nordenstadt, Germany) was incloneed for 15 minutes at 468705llowed by washing steps and staining with anti-R&D IgG-PE (minimal x reactivity; BioLegend).

Then sections were fixed in 70% ethanol (Hoechst 33342 is lost at this step) and stained for pimonidazole using the FITC-conjugated anti-pimonidazole monoclonal antibody (1 : 1000 dilution in PST).

Four high power fields (400x) were evaluated for percentage positive cells (0 100 % in 5%% steps), and staining intensity (0 to 3).

Paraffin-embedded sections were cut into 6 μm sections with 150 μm between steps, and were stained with hematoxylin and eosin (H&E).

Multiple step sections were made and stained with haematoxylin and eosin.

In the next step, cells were washed, fixed and stained with DAPI as described before.

Cryostat sections (6 μm) spanning the whole lesion area were prepared, fixed in −20 °C acetone and stained using a three-step immunoperoxidase staining kit (Vectastain).